The mechanism of the action of antigen specific suppressor factors (SF, secreted in vitro by specific Ly 2 positive 3 positive Ia (I-J) positive suppressor cells) was investigated in in vitro systems. The target cells of the suppressor factor is a Ly1 positive, nylon wool nonadherent cell within the helper cell pool. The action of in vitro secreted antigen specific suppressor factors is not genetically restricted: with antigens under Ir-gene control both responder and nonresponder mice produced specific SF, and SF act across H-2 barriers. Serological analysis of the SF revealed determinants shared by all antigen specific suppressor factors (but not helper factors) regardless of the specificity and species of origin, "constant" region determinants, as well as determinants characteristic to a given antigen combining site specificity and strain of origin, "variable" region or "idiotypic" determinants shared by helper and suppressor factors of the same specificity. Anti-constant region antibodies also abolished the function of antigen specific suppressor factors of human and monkey origin suggesting shared (or cross-reacting) determinants conserved in one functional class of T cell factors. The production of hybrids between antigen specific suppressor cells and a thymoma tumor line yielded clones continuously secreting antigen suppressor factors. On the basis of functional and serological analysis (I-J, constant and variable region determinants) the suppressor factors produced by hybrids resembled conventional SF on all aspects tested. Further biochemical analysis of specific factors secreted by hybrids is underway. Using methods well established in murine systems suppressor factors were also produced from human peripheral blood lymphocytes in vitro with several antigens. These factors (like factors produced in monkeys) act across species barriers and were tested using mouse spleen cells. They are antigen specific, seem to carry DR coded determinants and suppress both IgM and IgG responses. Further analysis of the human specific factors is in progress.